Eukaryotic DNA is compacted through the formation of a nucleoprotein complex known as chromatin. The primary protein components of this complex are the core histones H2A, H2B, H3 and H4. The core histones form an octameric complex around which DNA wraps to form the nucleosome which is the basic repeating structure that serves as the foundation for higher order chromatin folding. As the packaging of DNA into chromatin places severe constraints on the accessibility of DNA, the ability to regulate chromatin structure is critical for cellular processes that require access to DNA such as transcription, DNA replication and DNA repair. The post-translational modification of the core histones has emerged as an important mechanism by which chromatin structure is modulated in cells. Accordingly, the core histones are the sites of numerous modifications. An intriguing aspect of histones modifications, which has emerged in the past few years, is that they do not function in isolation. There are now several examples of cross-talk between different modifications. For example, the ubiquitylation of histone H2B lysine 123 is required for the methylation of histone H3 lysines 4 and 79. Hence, modifications on one histone can influence the presence of other modifications. Our proposal is to combine yeast genetics, stable isotope labeling and mass spectrometry to comprehensively and quantitatively identify the network of interactions that exist between histone modifications. We will systematically mutate all sites of histone modification in the yeast core histones. We will then use stable isotope labeling and mass spectrometry to quantitate all of the histone modifications in the mutant strain relative to a wild type control. In this way, an unbiased picture will be obtained of all the cross-talk that exists between modifications in the core histones. PUBLIC HEALTH RELEVANCE: Due to its enormous linear length, the genomic DNA of eukaryotes needs to be highly condensed to fit inside cells. The DNA is condensed through packaging with proteins known as histones into a complex called chromatin. Cells must also be able to regulate the degree to which specific regions of DNA are condensed so that the DNA can become accessible when necessary. One important mechanism to regulate chromatin condensation is through modifications to the histone proteins. This proposal seeks to identify interactions that exist between different sites of histone modification. An understanding of how histone modifications function is important for human health as defects in the regulation of histone modifications and chromatin structure play critical roles in diseases such as cancer.